We will use photoaffinity labeling as a means of identifying by electron microscopic autoradiography and cell fractionation experiments the receptor sites for ethidium in trypanosomes. Radiolabeled ethidium mono- and diazide will be synthesized, mixed with sensitive and resistant parasites (Trypanosoma brucei, lewisi, cruzi) and photolyzed to form covalent drug adducts from reversible drug complexes. Initial experiments have revealed trypnocidal action of ethidium monoazide in the dark enhancement in light. The efficacy, compared with ethidium bromide, of the new drugs will be determined in dark and following photolysis; and timing of the photolytic step will define the rates for drug penetration and binding. Competitive studies with parent drug will demonstrate the relevance of the photolytically labeled sites to authentic ethidium binding sites. Studies with drug resistant parasites should give clues to mechanisms for drug resistance. Success in project should lead to unambiguous identification of intracellular drug binding sites and provide major progress in understanding mechanisms for antiparasitic drug action of ethidium; provide an exciting new approach to the study of other antiparasitic drugs; and help to define host parasite relationships.